The present invention, in some embodiments thereof, relates to a method of generating collagen fibers.
Collagen is the principal structural protein in the body and constitutes approximately one-third of the total body protein. It comprises most of the organic matter of the skin, tendons, bones and teeth and occurs as fibrous inclusions in most other body structures. Some of the properties of collagen are its high tensile strength; its ion exchanging ability, due in part to the binding of electrolytes, metabolites and drugs; its low antigenicity, due to masking of potential antigenic determinants by the helical structure, and its low extensibility, semipermeability, and solubility. Furthermore collagen is a natural substance for cell adhesion. These properties make this protein suitable for fabrication of bioremodelable research products and medical devices such as implantable prostheses, cell growth substrates, and cellular and acellular tissue constructs.
Naturally, collagen is secreted by cells as a long triple-helical monomer, which polymerizes spontaneously into fibrils and strands, which often have a preferential orientation essential to the function of tissues such as skin, bone and nerve.
The exact structure of the collagen fibril is still unknown, but increasingly detailed models are becoming available, emphasizing the relation between fibril structure and function. Current models hint at a semi-crystalline (liquid crystal like) structure, combining a highly ordered arrangement in the axial direction and a short-range liquid-like order in the lateral direction.
Collagen in its monomeric form is soluble in cold acidic pH (˜pH 2) solutions, and can be precipitated in the form of fibrils by neutralizing the pH, increasing the temperature and/or the ionic strength. Fibrillogenesis is entropy driven—the loss of water molecules from monomer surfaces drives the collagen monomers out of solution and into assemblies with a circular cross-section, so as to minimize surface area.
The fibrils formed in-vitro display D-banding pattern of 67 nm wide cross striations typical of natural collagen fibrils formed in-vivo, but lack altogether the macroscopic order that is the basis of structural tissues. Fibrils precipitated out of bulk solutions form an entangled mesh reminiscent of spaghetti and not the neatly ordered arrays of fibrils observed in nature.
Collagen can be deposited from solution by a variety of processes including casting, lyophilization, electrospinning and other processes well known to one skilled in the art. In most of these procedures, collagen fibers of widely varying diameters and lengths from the micrometer range typical of conventional fibers down to the nanometer range are formed. Owing to their small diameters, electrospun fibers possess very high surface-to-area ratios and are expected to display morphologies and material properties very different from their conventional counterparts occurring in nature.
Numerous attempts to direct or align collagen fibrils for manufacturing of collagen matrices have been performed, employing various methods. Major efforts are aimed at creating 2D (collagen surface) or 3D (collagen scaffold) matrices. Exemplary methods include: alignment by surface templating, chemical patterning, nanolithography, electrochemical fabrication, use of a magnetic field, and by shear flow.
In vitro, collagen displays mesophase (liquid crystalline) properties at concentrations above ˜20 mg/ml (depending on acid concentration of the solvent). At concentrations between ˜20 to 50 mg/ml diffuse nematic phases appear in the bulk isotropic solution, observed as birefringent flakes. When the collagen concentration is increased, precholesteric patterns form—observed as spherulites, bands, or zigzag extinction patterns. Further increase in the concentration leads to formation of cholesteric patterns that become more and more compact until the entire sample displays characteristic fingerprint pattern.
At concentrations above 150 mg/ml, collagen fibrillar aggregates start to appear even in acidic solution, displaying the 67 nm banding typical of collagen fibrils, in a process reminiscent of a cholesteric-to-smectic (N*/SmA) transition.
U.S. Pat. No. 7,057,023 teaches spinning of liquid crystalline silk to generate silk fibers.
U.S. Patent Application No. 20070187862 teaches spinning a solution of liquid crystalline silk, wherein the solution is devoid of organic solvents to generate silk fibers.
U.S. Patent Application No. 20090069893 teaches formation of oriented collagen based materials from mesophase collagen by application of a shear force.
WO2011/064773 teaches generation of fibers from a solution of mesophase collagen.
Yaari et al., TISSUE ENGINEERING: Part A, Volume 19, Numbers 13 and 14, 2013, pages 1502-1505 teaches generation of fibers from a solution of mesophase collagen.
Additional background art includes Yaari et al., ACS Biomaterials Sci. Eng. Pages 349-360, Feb. 15, 2016.